Method for the determination of cystosine methylation in CpG islands

ABSTRACT

A method for the detection of cytosine methylation in DNA samples is described. First, DNA is extracted from a sample and bound to a surface. In the second step, a genomic DNA sample is preferably treated with a bisulfite (=disulfite, hydrogen sulfite), such that all unmethylated cytosine bases are converted to uracil, while the 5-methylcytosine bases remain unchanged. In the third step of the method, one or more oligonucleotides is (are) hybridized to the treated DNA as primers. In the fourth step of the method, the hybridized primer(s) is or are elongated in a polymerase reaction. Here, labeled guanine nucleotides are preferably utilized which are essentially incorporated only if cytosine bases were still present in the treated DNA. Consequently, the extent of incorporation of guanine bases and thus also the number of incorporated labels is proportional to the methylation in the DNA sample under investigation. In the fifth step of the method, the labeled nucleotides that were not incorporated in the polymerase reaction are removed. In the sixth step of the method, the number of labels in the fragment generated by the primer extension is approximately determined by directly or indirectly measuring signal intensities emitted by these labels.

The present invention concerns a method for the particularly sensitivedetection of cytosine methylation in DNA samples.

The levels of observation that have been well studied in molecularbiology according to developments in methods in recent years include thegenes themselves, the transcription of these genes into RNA and thetranslation to proteins therefrom. During the course of development ofan individual, which gene is turned on and how the activation andinhibition of certain genes in certain cells and tissues are controlledcan be correlated with the extent and nature of the methylation of thegenes or of the genome. In this regard, pathogenic states are alsoexpressed by a modified methylation pattern of individual genes or ofthe genome.

5-Methylcytosine is the most frequent covalently modified base in theDNA of eukaryotic cells. For example, it plays a role in the regulationof transcription, in genetic imprinting and in tumorigenesis. Theidentification of 5-methylcytosine as a component of genetic informationis thus of considerable interest. 5-Methylcytosine positions, however,cannot be identified by sequencing, since 5-methylcytosine has the samebase-pairing behavior as cytosine. In addition, in the case of a PCRamplification, the epigenetic information which is borne by the5-methylcytosines is completely lost.

A relatively new method that in the meantime has become the most widelyused method for investigating DNA for 5-methylcytosine is based on thespecific reaction of bisulfite with cytosine, which, after subsequentalkaline hydrolysis, is then converted to uracil, which corresponds inits base-pairing behavior to thymidine. In contrast, 5-methylcytosine isnot modified under these conditions. Thus, the original DNA is convertedso that methylcytosine, which originally cannot be distinguished fromcytosine by its hybridization behavior, can now be detected by“standard” molecular biology techniques as the only remaining cytosine,for example, by amplification and hybridization or sequencing. All ofthese techniques are based on base pairing, which is now fully utilized.The prior art, which concerns sensitivity, is defined by a method thatincorporates the DNA to be investigated in an agarose matrix, so thatthe diffusion and renaturation of the DNA is prevented (bisulfite reactsonly on single-stranded DNA) and all precipitation and purificationsteps are replaced by rapid dialysis (Olek A, Oswald J, Walter J. Amodified and improved method for bisulphite based cytosine methylationanalysis. Nucleic Acids Res. 1996 Dec. 15; 24(24):5064-6). Individualcells can be investigated by this method, which illustrates thepotential of the method. Of course, up until now, only individualregions of up to approximately 3000 base pairs long have beeninvestigated; a global investigation of cells for thousands of possiblemethylation analyses is not possible. Of course, this method also cannotreliably analyze very small fragments of small quantities of sample.These are lost despite the protection from diffusion through the matrix.

An overview of other known possibilities for detecting 5-methylcytosinescan be derived from the following review article: Rein T, DePamphilis ML, Zorbas H. Identifying 5-methylcytosine and related modifications inDNA genomes. Nucleic Acids Res. 1998 May 15; 26(10):2255-64.

The bisulfite technique has been previously applied only in research,with a few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Dörfler W,Horsthemke B. A single-tube PCR test for the diagnosis of Angelman andPrader-Willi syndrome based an allelic methylation differences at theSNRPN locus. Eur J Hum Genet. 1997 March-April; 5(2):94-8). However,short, specific segments of a known gene have always been amplifiedafter a bisulfite treatment and either completely sequenced (Olek A,Walter J. The pre-implantation ontogeny of the H19 methylation imprint.Nat Genet. 1997 November; 17(3):275-6) or individual cytosine positionshave been detected by a “primer extension reaction” (Gonzalgo M L, JonesP A. Rapid quantitation of methylation differences at specific sitesusing methylation-sensitive single nucleotide primer extension(Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15; 25(12):2529-31, WO-Patent9500669) or an enzyme cleavage (Xiong Z, Laird P W COBRA: a sensitiveand quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun. 15;25(12):2532-4). Detection has also been described by hybridization (Oleket al., WO 99/28498).

Urea improves the efficiency of bisulfite treatment prior to thesequencing of 5-methylcytosine in genomic DNA (Paulin R, Grigg G W,Davey M W, Piper A A. Urea improves efficiency of bisulphite-mediatedsequencing of 5′-methylcytosine in genomic DNA. Nucleic Acids Res. 1998Nov. 1; 26(21):5009-10).

Other publications which are concerned with the application of thebisulfite technique for the detection of methylation in the case ofindividual genes are:

Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA.Bioessays. 1994 June; 16(6):431-6, 431; Zeschnigk M, Schmitz B, DittrichB, Buiting K, Horsthemke B, Dörfler W. Imprinted segments in the humangenome: different DNA methylation patterns in the Prader-Willi/Angelmansyndrome region as determined by the genomic sequencing method. Hum Mol.Genet. 1997 March; 6(3):387-95; Feil R, Charlton J, Bird A P, Walter J,Reik W. Methylation analysis on individual chromosomes: improvedprotocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb.25; 22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R.Genomic sequencing indicates a correlation between DNA hypomethylationin the 5′ region of the pS2 gene and in its expression in human breastcancer cell lines. Gene. 1995 May 19; 157(1-2):261-4; WO 97-46705, WO95-15373 and WO-45560.

Another known method is the so-called methylation-sensitive PCR (HermanJ G, Graff J R, Myohanen S, Nelkin B D, Baylin S B (1996),Methylation-specific PCR: a novel PCR assay for methylation status ofCpG islands. Proc Natl Acad Sci USA. September 3; 93(18):9821-6). Forthis method, primers are used which hybridize either only to a sequencethat forms by the bisulfite treatment of a DNA which is unmethylated atthe respective position, or, vice versa, primers which bind only to anucleic acid which forms by the bisulfite treatment of a DNA methylatedat the respective position. Amplified products can be producedaccordingly with these primers, the detection of which in turn suppliesindications of the presence of a methylated or unmethylated position inthe sample to which the primers bind.

A newer method is also the detection of cytosine methylation by means ofa Taqman PCR, which has become known as “methyl light” (WO 00/70090). Itis possible with this method to detect the methylation state ofindividual positions or a few positions directly in the course of thePCR, so that a subsequent analysis of the products becomes superfluous.

An overview of the prior art in oligomer array production can be derivedalso from a special issue of Nature Genetics which appeared in January1999 (Nature Genetics Supplement, Volume 21, January 1999), theliterature cited therein and U.S. Pat. No. 5,994,065 on methods for theproduction of solid supports for target molecules such asoligonucleotides in the case of reduced nonspecific background signal.

Probes with multiple fluorescent labels have been used for scanning animmobilized DNA array. Particularly suitable for fluorescent labels isthe simple introduction of Cy3 and Cy5 dyes at the 5′-OH of therespective probe. The fluorescence of the hybridized probes is detected,for example, by means of a confocal microscope. The dyes Cy3 and Cy5,among many others, are commercially available.

Matrix-assisted laser desorptions/ionization mass spectrometry(MALDI-TOF) is a very powerful development for the analysis ofbiomolecules (Karas M, Hillenkamp F. Laser desorption ionization ofproteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988Oct. 15; 60(20):2299-301). An analyte is embedded in a light-absorbingmatrix. The matrix is vaporized by a short laser pulse and the analytemolecule is transported unfragmented into the gaseous phase. The analyteis ionized by collisions with matrix molecules. An applied voltageaccelerates the ions in a field-free flight tube. Ions are acceleratedto varying degrees based on their different masses. Smaller ions reachthe detector sooner than large ions.

MALDI-TOF spectroscopy is excellently suitable for the analysis ofpeptides and proteins. The analysis of nucleic acids is somewhat moredifficult (Gut, I. G. and Beck, S. (1995), DNA and Matrix Assisted LaserDesorption Ionization Mass Spectrometry. Molecular Biology: CurrentInnovations and Future Trends 1: 147-157.) For nucleic acids, thesensitivity is approximately 100 times poorer than for peptides anddecreases overproportionally with increasing fragment size. For nucleicacids, which have a backbone with a multiple negative charge, theionization process through the matrix is basically inefficient. InMALDI-TOF spectroscopy, the choice of matrix plays an imminentlyimportant role. Several very powerful matrices, which produce a veryfine crystallization, have been found for the desorption of peptides. Inthe meantime, several effective matrices have been developed for DNA,but the difference in sensitivity has not been reduced thereby. Thedifference in sensitivity can be reduced by modifying the DNA chemicallyin such a way that it resembles a peptide. Phosphorothioate nucleicacids, in which the usual phosphates of the backbone are substituted bythiophosphates, can be converted by simple alkylation chemistry into acharge-neutral DNA (Gut, I. G. and Beck, S. (1995), A procedure forselective DNA alkylation and detection by mass spectrometry. NucleicAcids Res. 23: 1367-1373). The coupling of a “charge tag” to thismodified DNA results in an increase in sensitivity by the same amount asis found for peptides. Another advantage of “charge tagging” is theincreased stability of the analysis in the presence of impurities, whichmake the detection of unmodified substrates very difficult.

Genomic DNA is obtained from DNA of cells, tissue or other assay samplesby standard methods. This standard methodology is found in referencessuch as Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual,1989.

After the invention of PCR, numerous variants became known in thefollowing few years, which refine this technique for the amplificationof DNA. In particular, multiplexing of the PCR (multiplex PCR) should bementioned here, in which more than 2 specific primers are used and thusa plurality of different, specific amplifications can be produced in onereaction vessel. Particularly interesting also is the so-called nestedPCR, which is used among other things for the detection of particularlysmall DNA quantities. This type of PCR is comprised of two successiveamplifications, wherein the primers of the second amplification liewithin the first amplificate and are not identical with the primers ofthe first amplification. In this way, a particular specificity isachieved, since the primers of the second amplification only function ifthe intended fragment was produced in the first amplification. Incontrast, the propagation of any possible byproducts of the firstamplification is excluded in the second amplification as much aspossible.

Accordingly, a great many methods for methylation analysis are priorart. The present invention, however, will provide a possibility for theanalysis of the degree of methylation overall in a CpG island.Preferably, a polymerase reaction, which facilitates conducting themethod, need not be conducted. It is essential within the framework of amethylation analysis in the field of clinical diagnosis that results ofinvestigation can be made available as rapidly as possible and that theexperimental expenditure is kept as small as possible. The methoddescribed here, which measures the extent of methylation overall in aCpG island, is particularly suitable for this purpose. In contrast tothe methods for methylation analysis that have been previouslydescribed, the methyllation state of an individual or several individualCpG positions is thus not determined. In many cases, the latter can be adisadvantage, since entire promotor regions can be present comethylated,i.e., many sequential CpG positions possess the same methylation state.

The present invention takes advantage of the fact that this methylationstate is assumed to be similar in numerous positions to be investigated,and a signal can be generated, which results from the sum of theseindividual positions. This makes possible a sensitivity which can besufficient without conducting a PCR reaction.

The method also takes advantage of the fact that guanine bases inbisulfite-treated DNA on one strand will only be incorporated in asubsequent polymerase reaction if a methylated cytosine was present inthe corresponding genomic DNA sample. If the sample DNA contains nomethylations at the respective positions, then guanine is notincorporated in the polymerase reaction.

And vice versa, also if a counterstrand was produced in a PCR reactionrelative to bisulfite-treated DNA (after bisulfite treatment, the DNAstrands of the sample are no longer complementary as they wereoriginally), then with this counterstrand as a template, a cytosine isincorporated in a subsequent polymerase reaction, only if originally amethylated cytosine was present in the corresponding genomic DNA sample.

It thus follows that guanines or cytosines will be incorporated only ifa methylation was present in the genomic DNA sample. The extent ofincorporation of guanines or cytosines (depending on the method eachtime; see above) is directly correlated with the extent of methylationin the investigated genomic DNA segment.

The method according to the invention thus consists of the followingsteps:

First, the genomic DNA is extracted from a sample and then preferablybound to a surface, this binding most preferably produced byhybridization to an immobilized oligonucleotide and in turn the genomicDNA is most preferably cleaved by restriction enzymes prior to thebinding.

It is preferred according to the invention that the DNA samples areobtained from serum or other body fluids of an individual.

It is additionally preferred according to the invention, that the DNAsamples are obtained from cell lines, blood, sputum, stool, urine,serum, cerebrospinal fluid, tissue embedded in paraffin, for example,tissue from intestine, kidney, brain, heart, prostate, lungs, eyes,breast or liver, histological slides and all possible combinationsthereof.

The enzymatic step for the DNA is most preferably conducted with arestriction endonuclease or several different restriction enzymes. Ifseveral restriction endonucleases are used, then it depends on therespective buffers whether these are applied sequentially orsimultaneously. The use of restriction enzymes according to theprotocols supplied by the manufacturers is known to the person skilledin the art.

In the second step, a genomic DNA sample is preferably treated with abisulfite (=disulfite, hydrogen sulfite), such that all unmethylatedcytosine bases are converted to uracil, while the 5-methylcytosine basesremain unchanged. Most preferably, this occurs at the surface onto whichthe sample DNA was already bound in the first step. It is mostparticularly preferred according to the invention that the chemicaltreatment is conducted with a bisulfite (=disulfite, hydrogen sulfite).It is also preferred that the chemical treatment is conducted afterembedding the DNA in agarose. It is also and additionally preferred thatin the chemical treatment, a reagent that denatures the DNA duplexand/or a radical trap are present.

In the third step of the method, one or more oligonucleotides is/arehybridized as primers to the treated DNA.

In the fourth step of the method, the hybridized primer(s) is or areelongated in a polymerase reaction. Here, labeled guanine nucleotidesare utilized which are essentially incorporated only if cytosine baseswere still present in the treated DNA. Consequently, the extent ofincorporation of guanine bases and thus also the number of incorporatedlabels is proportional to the methylation in the DNA sample underinvestigation. Most preferably, the polymerase reaction terminates atthe position which was cleaved in the first step by the use of arestriction endonuclease.

In the fifth step of the method, the labeled nucleotides that were notincorporated in the polymerase reaction are removed. Most preferablythis is done by simple washing steps in the case where the DNA ispresent bound to a surface.

In the sixth step of the method, the number of labels in the fragmentgenerated by primer extension is approximately determined by directly orindirectly measuring the signal intensity emitted by these labels.

The methylation state of the DNA sample in the investigated fragment isconcluded from the signal intensity.

Labels can be, for example, fluorescent labels, radionuclides, orremovable mass labels, which are detected in a mass spectrometer. Labelssuch as peptides, which are detected indirectly by the binding of anantibody which is labeled in a different way, however, can also be used.Chemical labels are also conceivable, which can be made visible only bysubsequent reaction with a marker molecule which is labeled in adifferent way and which can be, for example, a fluorescent dye. A greatmany possibilities for providing molecules with labels are familiar tothe person skilled in the art. The possibilities listed here will thusbe understood as examples, and other possibilities for labeling that arefamiliar to the person skilled in the art will be considered as acomponent of this invention.

In the above-named steps, the amplification of the treated DNA sample isdispensed with. The method is thus particularly applicable when thesample quantity is not limiting and the labels used can be detected withsufficient sensitivity. However, in the investigation of a CpG island,for example, according to the above-described method, since a pluralityof labels will be incorporated in the polymerase reaction, if the CpGisland was present methylated, a considerable increase in sensitivitywill be achieved also by the type of method conducted.

If an increased sensitivity is required due to a very small amount ofDNA, then the above-described method will be completed by a PCR reactionor another polymerase reaction, which is not just a linearly amplifyingreaction and which will be conducted after treatment according to thesecond method step. Preferably the (chemically) treated DNA sample willbe amplified with the use of preferably at least 2 primeroligonucleotides by means of a polymerase reaction, wherein preferably aheat-stable polymerase, nucleotides as well as a suitable reactionbuffer, as are often supplied with the polymerase and known to theperson skilled in the art, will be used.

It is also particularly preferred to conduct the amplifications ofseveral different fragments with more than 2 different primers in onereaction vessel and thus to carry out the amplification steps as amultiplex PCR. It is generally particularly preferred to conduct theamplifications as a polymerase chain reaction.

If, in addition, operation is also conducted with an additional PCR on asurface, then this is either a solid-phase PCR of the type in whichprimers are additionally bound to the surface for the PCR step, or afterthe PCR, there is a purification step, preferably by means of acommercial purification kit (such as, for example, from the companiesPromega or Qiagen) and subsequent binding of the PCR product to asurface onto which now the additional polymerase reaction forincorporating the labels will be conducted.

It is preferred that in the amplification, one of the primers is boundto a solid phase. For example, this solid phase can involvefunctionalized polymers, metals, glass or semiconductors such assilicon. The primers are bound preferably via bifunctional linkermolecules, which are bound to a silanized surface or, for example, viathioates in the primer or thiol modifications to bromacetyl [acetylbromide]-derivatized surfaces or gold.

In another, preferred alternative variant of the method, theincorporation of labels is produced also in a PCR reaction. In thiscase, the reaction occurs preferably without binding the primers to asolid phase. Instead, the PCR product is separated, for example, by gelelectrophoresis, from other by-products and educts. The intensity of thesignals emitted from the labels is determined by the bands or bandpatterns obtained and thus a conclusion is made of the degree ofmethylation in the fragment of sample DNA investigated.

If the complementary counterstrand to the treated DNA fragments is alsoproduced in an amplification, then in the case of treatment with abisulfite, in this counterstrand, adenine corresponds to an unmethylatedcytosine position and guanine corresponds to a methylated cytosineposition in the DNA sample. Therefore, it is also possible afteramplification to conduct the method logically also with a labeledcytosine.

The primers used in the polymerase reactions most preferably do notamplify fragments of genomic DNA that is not treated with bisulfite (oronly do so to a negligibly small extent), so that they are specific forthe DNA converted with bisulfite. This protects from erroneous resultsin the case of an incomplete conversion reaction with sodium bisulfite,for example.

It is further preferred that the analysis is conducted by means ofhybridization to oligomer arrays, wherein oligomers can be nucleic acidsor molecules such as PNAs that are similar in their hybridizationproperties.

It is preferred that the methylation state of more than 10 methylationpositions of the DNA to be analyzed is detected in one experiment.

It is also preferred according to the invention that the analysis isconducted by additionally measuring the length of the amplified DNAunder investigation, whereby methods for length measurement comprise gelelectrophoresis, capillary gel electrophoresis, chromatography (e.g.HPLC), mass spectrometry and other suitable methods. The fragments arethus detected via the labels incorporated in the polymerase reaction.

In addition, it is preferred that a conclusion is made on the presenceof a disease or another medical condition of the patient from themethylation degree of individual or several different CpG islandsinvestigated.

The labels are preferably introduced either by a label of thenucleotides during the polymerase reaction or amplification in theproduced labeled fragments.

In addition, it is particularly advantageous that the labels arefluorescent labels or/and that the labels are radionuclides or/and thatthe labels are removable mass labels, which are detected in a massspectrometer.

It is also preferred according to the invention that the fragments aredetected overall in the mass spectrometer and are thus clearlycharacterized by their mass. Therefore, each incorporated labelcontributes a specific mass for the methylation, so that the number ofmethylations in the CpG island can be concluded from the measuredmolecular mass.

Another subject of the present invention is also the use of a methodaccording to the invention for the diagnosis and/or prognosis of adverseevents for patients or individuals, whereby these adverse events belongto at least one of the following categories: undesired drug effects;cancer diseases; CNS malfunctions, damage or disease; symptoms ofaggression or behavioral disturbances; clinical, psychological andsocial consequences of brain damage; psychotic disturbances andpersonality disorders; dementia and/or associated syndromes;cardiovascular disease, malfunction and damage; malfunction, damage ordisease of the gastrointestinal tract; malfunction, damage or disease ofthe respiratory system; lesion, inflammation, infection, immunity and/orconvalescence; malfunction, damage or disease of the body as [aconsequence of] an abnormality in the development process; malfunction,damage or disease of the skin, the muscles, the connective tissue or thebones; endocrine and metabolic malfunction, damage or disease; headachesor sexual malfunction.

The use of a method according to the invention is thus advantageous fordistinguishing cell types or tissues or for investigating celldifferentiation.

The subject of the present invention is also a kit comprised of areagent containing bisulfite, primers for the polymerase reaction, aswell as, optionally, instructions for conducting an assay according tothe invention. A microtiter plate which possesses an activated surfacefor the immobilization of sample DNA and in which subsequent reactionsteps also can be carried out is additionally a preferred component ofthis kit.

The method according to the invention consequently consists of thefollowing steps:

a) DNA is extracted from a sample,

b) the DNA is treated, preferably with a bisulfite (=disulfite, hydrogensulfite), in such a way that cytosine is converted into a base that isdifferent in its base pairing behavior in the DNA duplex, while5-methylcytosine remains unchanged,

c) one or more oligonucleotide primers are hybridized to the treatedDNA,

d) the hybridized primers are extended in a polymerase reaction whereinlabeled nucleotides are essentially incorporated only if cytosine baseswere still present in the treated DNA after step b) and wherein theextent of the incorporation of labeled nucleotides correlates with themethylation in the DNA sample under investigation,e) the labeled nucleotides that were not incorporated in the polymerasereaction are removed,f) the number of labels in the fragment generated by primer extension isapproximately determined by measuring the signal intensity emitted bythese labels.

It is particularly preferred that the sample DNA is obtained from serumor other body fluids of an individual.

It is particularly preferred that the sample DNA is obtained from celllines, blood, sputum, stool, urine, serum, cerebrospinal fluid, tissueembedded in paraffin, for example, tissue from eyes, intestine, kidney,brain, heart, prostate, lungs, breast or liver, histological slides andall possible combinations thereof.

It is also particularly preferred that the treatment according to claim1b) is conducted with a bisulfite (=disulfite, hydrogen sulfite). It isalso particularly preferred that the chemical treatment is conductedafter embedding the DNA in agarose or most preferably after binding theDNA to a surface. In another particularly preferred method variant, inthe chemical treatment, a reagent that denatures the DNA duplex and/or aradical trap is present.

It is also particularly preferred that genomic DNA is extracted from asample and then is bound to a surface. It is also particularly preferredthat this binding is produced by hybridization to an immobilizedoligonucleotide. It is also preferred that the extracted DNA is cleavedby restriction enzymes prior to the binding.

In a particularly preferred conducting of the method, several differentoligonucleotides are hybridized as primers to the treated DNA.

The labeled nucleotides are most preferably guanine derivatives, whereinthese are essentially incorporated in the polymerase reaction, only if acytosine methylation was present in the DNA sample at the correspondingpositions. It is also preferred that the extent of incorporation ofguanine bases and thus also the number of incorporated labels isproportional to the methylation in the DNA sample under investigation.

In a particularly preferred variant of the method, the polymerasereaction terminates preferably at the position which was cleaved by theuse of a restriction endonuclease prior to the DNA isolation.

In another preferred method variant, the labeled nucleotides notincorporated in the polymerase reaction are removed by washing steps andthe DNA is present bound to a surface.

The labels are, preferably, fluorescent labels, radionuclides,chemiluminescent labels or removable mass labels, which are detected ina mass spectrometer. It is also preferred that the labels are detectedindirectly by the binding of an antibody labeled in a different way.

In another particularly preferred variant of the method, the treated DNAsample is amplified with the use preferably of at least two primeroligonucleotides, preferably by means of a polymerase chain reaction. Inanother particularly preferred method variant, the labeled nucleotidesare cytosine derivatives, wherein these are essentially incorporated inthe polymerase reaction, only if a cytosine methylation was present inthe DNA sample at the corresponding positions.

A method in which the amplification of several fragments is conducted inone reaction vessel in the form of a multiplex-PCR is also preferred.

In another particularly preferred variant of the method, at least in oneof the amplifications, one of the respective primers is bound to a solidphase.

The method according to one of the above, further characterized in thatthe amplificates are detected as a whole in the mass spectrometer andare thus clearly characterized by their mass.

The subject of the present invention is also the use of one of thedescribed methods for the diagnosis and/or prognosis of adverse eventsfor patients or individuals, whereby these adverse events belong to atleast one of the following categories: undesired drug effects; cancerdiseases; CNS malfunctions, damage or disease; symptoms of aggression orbehavioral disturbances; clinical, psychological and social consequencesof brain damage; psychotic disturbances and personality disorders;dementia and/or associated syndromes; cardiovascular disease,malfunction and damage; malfunction, damage or disease of thegastrointestinal tract; malfunction, damage or disease of therespiratory system; lesion, inflammation, infection, immunity and/orconvalescence; malfunction, damage or disease of the body as [aconsequence of] an abnormality in the development process; malfunction,damage or disease of the skin, the muscles, the connective tissue or thebones; endocrine and metabolic malfunction, damage or disease; headachesor sexual malfunction.

The subject of the invention is also the use of a method according toone of the preceding claims for distinguishing cell types or tissues orfor investigating cell differentiation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a particularly preferred variant of the invention:

a) enzymatically cleaved sample DNA is bound to a surface and thus isseparated from accompanying material

b) the DNA bound to the surface is denatured and then, for example,chemically treated with a bisulfite

c) a primer is bound to the DNA

d) an enzymatic primer extension reaction (nucleotides represented asdashed lines: - - - ) is conducted, and labels are incorporated only ifcytosine methylations (*) were present previously in the sample DNA atthe referred position.

e) the remaining labeled nucleotides and reaction components are removedin one washing step

f) the intensity of fluorescence emitted from the incorporated label ismeasured.

1. A method for the detection of cytosine methylation at more than 10methylation positions within a CpG island in a DNA sample comprising: a)extracting DNA from a sample, b) binding the extracted DNA to a surface,c) treating the bound DNA in such a way that cytosine is converted intoa base that is different in its base pairing behavior in the DNA duplex,while 5-methylcytosine remains unchanged, d) hybridizing oneoligonucleotide primer to the treated DNA, e) extending the hybridizedprimer by more than one nucleotide in a polymerase reaction to determinethe methylation state at more than 10 methylation positions within a CpGisland, wherein labeled guanine derivatives are essentially incorporatedonly if cytosine bases were still present in the DNA treated accordingto step c) and wherein the extent of the incorporation of labelednucleotides correlates with the methylation of the CpG island in the DNAsample under investigation, f) removing the labeled nucleotides thatwere not incorporated in the polymerase reaction, g) determiningapproximately the number of labels in the fragment generated by primerextension by measuring the signal intensity emitted by these labels todetermine the methylation state at more than 10 methylation positions,wherein the detection of more than 10 cytosine methylation positionswithin said CpG island in the DNA sample is afforded.
 2. The methodaccording to claim 1, further characterized in that the DNA samples areobtained from serum or other body fluids of an individual.
 3. The methodaccording to claim 1, further characterized in that the DNA samples areobtained from cell lines, blood, sputum, stool, urine, serum,cerebrospinal fluid, tissue embedded in paraffin, for example, tissuefrom eyes, intestine, kidney, brain, heart, prostate, lungs, breast orliver, histological slides and all possible combinations thereof.
 4. Themethod according to claim 1, further characterized in that the treatmentaccording to claim 1c) is conducted with a bisulfite.
 5. The methodaccording to claim 4, further characterized in that the chemicaltreatment is conducted after embedding the DNA in agarose.
 6. The methodaccording to claim 4, further characterized in that in the chemicaltreatment, a reagent that denatures the DNA duplex and/or a radical trapis present.
 7. The method according to claim 1, wherein the binding ofthe extracted DNA to a surface comprises binding the DNA to animmobilized oligonucleotide by hybridization.
 8. The method according toclaim 1, further characterized in that the extracted DNA is cleaved byrestriction enzymes prior to the binding.
 9. The method according toclaim 1, further characterized in that the polymerase reactionterminates preferably at the position which was cleaved by the use of arestriction endonuclease prior to the DNA isolation.
 10. The methodaccording to claim 1, further characterized in that the labelednucleotides not incorporated in the polymerase reaction are removed bywashing steps and the DNA is present bound to a surface.
 11. The methodaccording to claim 1, further characterized in that the labels arefluorescent labels, radionuclides, chemiluminescent labels or removablemass labels, which are detected in a mass spectrometer.
 12. The methodaccording to claim 1, further characterized in that the labels areindirectly detected by the binding of an antibody labeled in a differentway.
 13. The method according to claim 1, further characterized in thatthe amplificates are detected are a whole in a mass spectrometer and arethus clearly characterized by their mass.
 14. The method according toclaim 1, wherein said treating step comprises treating the DNA with adisulfite.
 15. The method according to claim 1, wherein said treatingstep comprises treating the DNA with hydrogen sulfite.
 16. A method forthe diagnosis and/or prognosis of adverse events for patients orindividuals, whereby these adverse events belong to at least one of thefollowing categories: undesired drug effects; cancer diseases; CNSmalfunctions, damage or disease; symptoms of aggression or behavioraldisturbances; clinical, psychological and social consequences of braindamage; psychotic disturbances and personality disorders; dementiaand/or associated syndromes; cardiovascular disease, malfunction anddamage; malfunction, damage or disease of the gastrointestinal tract;damage or disease of the respiratory system; lesion, inflammation,infection, immunity and/or convalescence; malfunction, damage or diseaseof the body as an abnormality in the development process; malfunction,damage or disease of the skin, the muscles, the connective tissue or thebones; endocrine and metabolic malfunction, damage or disease; headachesor sexual malfunction, said method comprising: a) extracting DNA from asample, b) binding the extracted DNA to a surface, c) treating the boundDNA in such a way that cytosine is converted into a base that isdifferent in its base pairing behaviour in the DNA duplex, while5-methylcytosine remains unchanged, d) hybridizing one oligonucleotideprimer to the treated DNA, e) extending the hybridized primer by morethan one nucleotide in a polymerase reaction to determine themethylation state at more than 10 methylation positions within a CpGisland, wherein labelled guanine derivatives are essentiallyincorporated only if cytosine bases were still present in the DNAtreated according to step c) and wherein the extent of the incorporationof labelled nucleotides correlates with the methylation of the CpGisland in the DNA sample under investigation, f) removing the labellednucleotides that were not incorporated in the polymerase reaction, g)determining approximately the number of labels in the fragment generatedby primer extension by measuring the signal intensity emitted by theselabels to determine the methylation state at more than 10 methylationpositions, wherein the detection of more than 10 cytosine methylationpositions within said CpG island in the DNA sample is afforded todiagnose or prognose said adverse events for patients or individuals.17. A method for the differentiation of cell types or tissues or for theinvestigation of cell differentiation, said method comprising: a)extracting DNA from a sample, b) binding the extracted DNA to a surface,c) treating the bound DNA in such a way that cytosine is converted intoa base that is different in its base pairing behaviour in the DNAduplex, while 5-methylcytosine remains unchanged, d) hybridizing oneoligonucleotide primer to the treated DNA, e) extending the hybridizedprimer by more than one nucleotide in a polymerase reaction to determinethe methylation state at more than 10 methylation positions within a CpGisland, wherein labelled guanine derivatives are essentiallyincorporated only if cytosine bases were still present in the DNAtreated according to step c) and wherein the extent of the incorporationof labelled nucleotides correlates with the methylation of the CpGisland in the DNA sample under investigation, f) removing the labellednucleotides that were not incorporated in the polymerase reaction, g)determining approximately the number of labels in the fragment generatedby primer extension by measuring the signal intensity emitted by theselabels to determine the methylation state at more than 10 methylationpositions, wherein the detection of more than 10 cytosine methylationpositions within said CpG island in the DNA sample is afforded todifferentiate cell types or tissues or for the investigation of celldifferentiation.